核桃多肽对过氧化氢及β淀粉样肽25-35诱导PC12细胞氧化损伤的影响
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摘要
目的:探讨核桃多肽对过氧化氢(H_2O_2)及β淀粉样肽25-35(Aβ25-35)导致的PC12细胞损伤的保护作用。方法:以H_2O_2及Aβ25-35损伤PC12细胞建立氧化应激损伤模型,以不同浓度核桃多肽(0.1,0.25,0.5,0.75,1,2 mg·L~(-1))预保护PC12细胞,采用MTT法检测细胞活力;采用乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GSH-Px)等试剂盒检测H_2O_2及Aβ25-35对PC12细胞的氧化损伤,并研究核桃多肽的抗氧化作用;采用DCFH-DA结合荧光分光光度计检测细胞内的活性氧(ROS)水平。结果:与模型组相比,核桃多肽呈剂量依赖性地提高H_2O_2及Aβ25-35诱导的PC12细胞存活率;核桃多肽组的抗氧化酶SOD、CAT和GSH-Px活性明显提高,MDA和细胞内的ROS水平明显降低(P<0.05或P<0.01)。结论:核桃多肽对H_2O_2及Aβ25-35导致的PC12细胞损伤具有良好的保护作用,其作用机制可能与提高PC12细胞抗氧化能力有关。
Objective: To investigate the protective effect of walnut peptide on the damage of PC12 cells induced by β-amyloid protein 25-35( Aβ25-35) and hydrogen peroxide( H_2O_2). Methods: PC12 cells were injured by H_2O_2 and Aβ25-35 to establish oxidative stress injury model and different concentrations of walnut polypeptide( 0. 1,0. 25,0. 5,0. 75,1 and 2 mg·L~(-1)) were administrated to provide pre-protection for PC12 cells. The cell viability was measured by MTT. Lactate dehydrogenase( LDH),superoxide dismutase( SOD),malondialdehyde( MDA),catalase( CAT) and glutathione peroxidase( GSH-Px) kits were used to detect the oxidative damage of H_2O_2 and Aβ25-35 on PC12 cells,and study the antioxidant effect of walnut polypeptide. The intracellular reactive oxygen species( ROS) levels were detected by DCFH-DA combined with a fluorescence spectrophotometer. Results: Compared with the model group,walnut polypeptide increased the survival rate of PC12 cells induced by H_2O_2 and Aβ25-35 in a dose-dependent manner,the activities of antioxidant enzymes SOD,CAT and GSH-Px in walnut polypeptide group were significantly increased,and the levels of MDA and ROS in cells were significantly increased( P < 0. 05 or P < 0. 01). Conclusion: Walnut polypeptide has a good protective effect on PC12 cells damaged by H_2O_2 and Aβ25-35,which may be related to its anti-oxidant action.
Objective: To investigate the protective effect of walnut peptide on the damage of PC12 cells induced by β-amyloid protein 25-35( Aβ25-35) and hydrogen peroxide( H_2O_2). Methods: PC12 cells were injured by H_2O_2 and Aβ25-35 to establish oxidative stress injury model and different concentrations of walnut polypeptide( 0. 1,0. 25,0. 5,0. 75,1 and 2 mg·L~(-1)) were administrated to provide pre-protection for PC12 cells. The cell viability was measured by MTT. Lactate dehydrogenase( LDH),superoxide dismutase( SOD),malondialdehyde( MDA),catalase( CAT) and glutathione peroxidase( GSH-Px) kits were used to detect the oxidative damage of H_2O_2 and Aβ25-35 on PC12 cells,and study the antioxidant effect of walnut polypeptide. The intracellular reactive oxygen species( ROS) levels were detected by DCFH-DA combined with a fluorescence spectrophotometer. Results: Compared with the model group,walnut polypeptide increased the survival rate of PC12 cells induced by H_2O_2 and Aβ25-35 in a dose-dependent manner,the activities of antioxidant enzymes SOD,CAT and GSH-Px in walnut polypeptide group were significantly increased,and the levels of MDA and ROS in cells were significantly increased( P < 0. 05 or P < 0. 01). Conclusion: Walnut polypeptide has a good protective effect on PC12 cells damaged by H_2O_2 and Aβ25-35,which may be related to its anti-oxidant action.
引文
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2 Vickers JC,Dickson TC,Adlard PA,et al.The cause of neuronal degeneration in Alzheimer's disease[J].Pro Neurobiol,2000,60(2):139-165
3 Butterfield DA,Drake J,Pocernich C,et al.Evidence of oxidative damage in Alzheimer's disease brain:central role for amyloid betapeptide[J].Trends Mol Med,2001,7(12):548-554
4 Bourdel-Marchasson I,Peuchant E,Richard-Harston S,et al.Antioxidant defences and oxidative stress markers in erythrocytes and plasma from normally nourished elderly Alzheimer patients[J].Age Ageing,2001,30(3):235-241
5 Martínez ML.Walnut(Juglans regia L.):genetic resources,chemistry,by-products[J].J Sci Food Agr,2010,90(12):1959-1967
6 明·李时珍.本草纲目[M].北京:人民卫生出版社,1978:1803
7 Sze-Tao K WC,Sathe SK.Walnuts(Juglans regia L.):proximate composition,protein solubility,protein amino acid composition and protein in vitro,digestibility[J].J Sci Food Agr,2000,80(9):1393-1401
8 Muthaiyah B,Essa MM,Lee M,et al.Dietary supplementation of walnuts improves memory deficits and learning skills in transgenic mouse model of Alzheimer's disease[J].J Alzheimers Dis,2014,42(2):1397-1405
9 Zou J,Cai PS,Xiong CM,et al.Neuroprotective effect of peptides extracted from walnut(Juglans Sigilata Dode)proteins on Aβ25-35-induced memory impairment in mice[J].华中科技大学学报(医学英德文版),2016,36(1):21-30
10 Shafer TJ,Atchison WD.Transmitter,ion channel and receptor properties of pheochromocytoma(PC12)cells:a model for neurotoxicological studies[J].Neurotoxicology,1991,12(3):473-492
11 邹娟.核桃多肽对体内外老年痴呆实验模型的干预作用研究[D].武汉:华中科技大学硕士学位论文,2016
12 赵保路.自由基、天然抗氧化剂与神经退行性疾病[J].生物物理学报,2010,26(4):263-274
13 李彩莲.小檗碱对H2O2和Aβ(25-35)诱导PC12细胞损伤的作用及其机制[D].武汉:华中科技大学硕士学位论文,2006
14 Rukhsana SD,Allan B.Role of oxidative stress in the progression of Alzheimer's disease[J].J Alzheimers Dis,2010,19(19):341-353
15 刘真真.苯海索对β-淀粉样蛋白25-35介导的PC12细胞损伤的保护作用及其机制研究[D].郑州:河南大学硕士学位论文,2009
16 Melo A,Monteiro L,Lima RMF,et al.Oxidative Stress in Neurodegenerative Diseases:Mechanisms and Therapeutic Perspectives[J].Oxid Med Cell Longev,2011,2011:467180
17 Mancuso M,Coppede F,Migliore L,et al.Mitochondrial dysfunction,oxidative stress and neurodegeneration[J].J Alzheimers Dis,2006,10(1):59-73
18 Yan SD,Yan SF,Chen X,et al.Non-enzymatically glycated tau in Alzheimer's disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloidβ-peptide[J].Nat Med,1995,1(7):693-699
19 Yan SD,Fu J,Soto C,Yan,S.D.et al.An intracellular protein that binds amyloid-peptide and mediates neurotoxicity in Alzheimer's disease[J].Nature,1997,389(6652):689-695
20 姜招峰,杨翰仪.氧自由基对CAT、SOD和GPX的氧化修饰研究[J].北京联合大学学报:自然科学版,2003,17(3):12-17
21 Gervais FG,Xu D,Robertson GS,et al.Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation[J].Cell,1999,97(3):395-406
22 Davies SM,Poljak A,Duncan MW,et al.Measurements of protein carbonyls,ortho-and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats[J].Free Radic Biol Med,2001,31(2):181-190